The analysis of DNA and/or RNA from bacterial cells and virus particles is a key step in many areas of technology such as, for example, diagnostics, environmental monitoring, forensics and molecular biology research. In order to analyze samples containing nucleic acids, it is usually necessary to carry out two procedures. Firstly, the sample is broken down, isolated and concentrated to produce a purified nucleic acid extract. Secondly, the purified nucleic acid extract is amplified in order increase the amount of nucleic acid present to facilitate detection of the nucleic acid.
Conventionally, extraction, purification and amplification of the nucleic acid is carried out manually in a laboratory by a trained technician. This not only requires the presence of a skilled user, it also leads to a significant error rate due to user errors. In addition, this conventional extraction requires the extraction, purification and amplification to take place away from the point-of-care and, as a result, the result of the biological assay is delayed. Therefore, there is a need for providing a biological assay that reduces and simplifies user input and allows the assay to be carried out when and where the sample is actually taken, for example within the doctor's surgery, the clinic, the veterinary surgery or even in the patient's home or in the field.
Microfabricated “lab-on-a-chip” devices are an attractive option for carrying out contained biological reactions. These devices require minimal reagent handling by the user and also permit the use of small sample volumes, a significant advantage for biological reactions which require expensive reagents.
One previous approach to providing a microfabricated “lab-on-a-chip” device to extract and purify a sample comprising a nucleic acid is described in WO 2005/073691. In this document, a sample containing cells and/or particles is filtered. The filtrate (i.e. the cells and/or particles) is subject to lysis by a lysis fluid. Then, the lysed sample is passed through a nucleic acid extraction unit. The nucleic acids are extracted and remain in the extraction unit whereas the lysis fluid passes through the unit. An example of a suitable nucleic acid extraction method involves the binding of DNA to silica particles in the presence of a chaotropic agent (see Boom et al, J. Clin. Microbiol. 1990, 28, 495-503). The extracted nucleic acids are washed with one or more washing solvents, followed by extraction of the nucleic acids with an eluant. This step also serves to concentrate the nucleic acid.
WO 2005/073691 describes how a single pump may be used to actuate all fluids within its system once the sample has been syringed into the system. WO 2005/073691 then describes one way of achieving this, namely to provide the lysis fluid, washing fluids and eluant in a single channel separated by air gaps.
Once the nucleic acid has been extracted, concentrated and purified, it is then usually necessary to amplify it. While conventionally the Polymerase Chain Reaction (PCR) technique is used, a different amplification technique that may be used in some circumstances is Nucleic Acid Sequence Based Amplification (NASBA). As will be appreciated by the person skilled in the art, NASBA is different from PCR in several ways. In particular, PCR involves thermal cycling of a sample that generally produces only DNA amplification products while NASBA is an isothermal technique that is generally used to produce RNA amplification products.
A microfabricated system that is especially designed for carrying out NASBA is described in WO 02/22265. This system comprises two chambers. The first chamber heats the sample up, denatures it and facilitates the binding of primers to the denatured sample. The second chamber contains the NASBA enzymes and heats the sample isothermally to a temperature of about 41° C.
In order to carry out amplification, it is necessary to mix the nucleic acid sample with primers. These primers require the presence of a mixing fluid. This fluid may comprise one or both of DMSO and sorbitol. In WO 02/22265, it is described how this fluid is pre-loaded into the first reaction chamber and the mixing of the sample with the fluid occurs within the first reaction chamber.